Development of optimal strategies to inhibit ERK signaling in tumors with RAF and MEK mutations Abstract Clinical tumor sequencing is increasingly being used in multiple cancer types to inform the care of individual cancer patients. However, only a small number of mutant alleles are currently used by physicians to guide treatment decisions, whereas most mutations, including those in well-studied cancer genes such as BRAF, remain of unknown biologic and clinical significance. The long-term objective of this proposal is to expand the clinical utility of sequencing by understanding the mechanisms of activation of the broader array of mutant alleles in the RAF and MEK kinases and their sensitivity to selective pharmacologic inhibitors of RAF, MEK, and ERK. By directly comparing the sensitivity of cellular models to patient responses, within the context of a co-clinical trial paradigm, we seek to expand the population of patients who benefit from treatment with ERK pathway inhibitors. Three specific aims are proposed. In Aim 1, we will biologically characterize BRAF, CRAF (RAF1), ARAF, MEK1, and MEK2 variants of unknown biologic and clinical significance identified in patients treated with RAF, MEK, and ERK inhibitors. Prioritization for detailed functional studies will be given to RAF or MEK mutant alleles of unknown clinical significance identified in patients treated with novel ERK pathway inhibitors within the context of clinical trials. Studies will be performed using engineered isogenic cell lines and patient-derived xenograft (PDX) and cell line models. In Aim 2, we will identify mechanisms of acquired resistance to RAF dimer inhibitors, a novel class of RAF inhibitors that has shown promising activity in preclinical models driven by non-V600 BRAF mutations, including BRAF fusions, which are intrinsically resistant to current FDA-approved RAF inhibitors such as vemurafenib. Specifically, we will utilize laboratory models selected for acquired resistance to RAF dimer inhibitors in parallel with pretreatment and disease- progression biopsies of patients treated with these agents to identify and functionally validate mechanisms of acquired resistance to RAF dimer inhibitors. Finally, in Aim 3, we will identify and validate mechanisms of resistance to MEK and ERK inhibitors in patients with activating mutations of MEK1 and MEK2. In both Aims 2 and 3, we will utilize a custom, next-generation sequencing platform designed to detect putative resistance mutations using cell-free DNA to define the timing at which such alterations arise during drug treatment. By distinguishing those RAF and MEK mutations that are biologically functional from those that are passenger mutations, and by defining the sensitivity of the former to FDA-approved and investigational targeted inhibitors of this pathway, the studies proposed here will expand the treatment options for this molecularly defined subset of cancer patients. The models of non-V600E BRAF-, A/CRAF-, or MEK1/2-mutant cancer developed here will also serve as a unique resource for future studies of novel RAF, MEK, and ERK inhibitors or novel drug combinations that could prevent or delay the emergence of treatment-resistant clones.